我们感谢基督教 Flierl, 卡嘉彼得, 克里斯蒂娜 Schnürer (所有分子和实验性手术的分裂), 赛门 Appelt, 迈克尔 Mroz (核心单位的资产管制系统分类) 和西蒙 Völkl (核心单位的资产管制局-Immunomonitoring), 为优秀的技术援助。这项工作得到了赠款的支持, 对大学医学中心埃尔兰根, 德国研究基金会 (DFG: 2438, SP2) 和鲁兹基金会, 由罗伯特-Pfleger-基金会 IZKF 的交叉学科中心的 EN/MS向 VSS 以及由德国研究基金会授予 MS 的赠款 [DFG: KFO257 (分项目 4), SFB 796 (分项目 B9)]。

隔离过程已被大学医学中心埃尔兰根 (#159_15 B, TuMiC 研究) 的地方伦理委员会批准。患者纳入标准如下: CRC, UICC 期 IV, 无炎症性肠病病史, 无新辅助治疗。
1. 单细胞隔离的手术和组织制备
<ol><li>从 CRC 患者 (癌组织 > 0.5 克, 中央非坏死性肿瘤) 手术获得标本; 正常结肠组织 > 10 厘米离肿瘤部位较远)。</li>
	<li>所获得的组织片主要 < 1 克为癌症, 但 > 1 g 为正常结肠。如果获得的碎片 > 1 克, 把它们分成多个 1 g 块使用新鲜的手术刀进一步组织处理。 注: 如果肿瘤组织片在发病时低于0.5 克, 隔离通常会失败。</li>
	<li>用40毫升的冰冷汉克斯平衡盐溶液 (HBSS) 在50毫升离心管中补充青霉素/链霉素/amphothericin B (1% HBSS 笔/链球菌/ampho), 用无菌钳收集新鲜未固定的组织件, 并迅速将其转移到实验室. 注意: 结肠组织经常受到细菌/真菌的高度污染, 从发病开始。在整个隔离过程中, 必须将笔/链球菌/ampho 添加到所有解决方案中, 以消除污染的有机体。</li>
	<li>用40毫升冰冷 HBSS 笔/链球菌/ampho (见步骤 1.3) 在50毫升离心管中, 用不育钳将组织件按顺序从一个离心管转移到下一管, 以4倍的时间冲洗每块纸巾。<ol>
<li>每步后清洁钳70% 乙醇。</li>
		<li>使用单独的镊子为癌症和正常结肠组织片。</li>
		<li>权衡所有的组织碎片。 注: 请勿将组织件直接放在秤上进行称量。洗涤后, 在进入组织片之前和之后, 重装含有组织的最后一个离心管。用减法计算单个组织块的重量。</li>
	</ol>
</li>
</ol>2. 单细胞悬浮液的产生
注意: 建议在任何时候保持组织湿润。
<ol><li>如果它存在, 去除附加的脂肪, 其他非肿瘤组织, 或潜在坏死的组织部分, 从手术标本转移的组织块在无菌细胞培养培养皿使用无菌钳。<ol>
<li>通过添加3-5 毫升的 HBSS/链球菌/ampho, 使组织片在任何时候都保持湿润。</li>
		<li>使用新鲜的无菌手术刀修剪组织件 (图 1)。咨询病理学家, 以防组织鉴定不明。</li>
	</ol>
</li>
	<li>将剩余的组织切成小块 (大约 2 x 2 x 2 毫米3), 使用带有弯曲刀片的新手术刀。将带无菌钳的组织件转移到组织离解管 (例如, gentleMACS C 管) 预填充前预热 (37 °c) 细胞培养培养基 (Dulbecco´s 改性鹰培养基 [DMEM] 基介质) 根据制造商的说明. 注意: 试着像摇摆工具一样使用手术刀。不要挤压细胞以避免组织损伤。</li>
	<li>使用组织 dissociator (如 gentleMACS 奥克托 dissociator) 与人体组织的肿瘤离解试剂盒结合使用 "37C_h_TDK_1" 的 manufacturer´s 指示的程序, 消化/游离组织件。</li>
	<li>离心分离管的1分钟在 300 x 克室温 (RT)。</li>
	<li>添加10毫升预热 (37 °c) DMEM 辅以0.5% 胎牛血清 (DMEM 低) 到每管使用用细胞过滤器 (100 µm 孔径) 对50毫升离心管顶部的细胞悬浮液进行血清学吸管和过滤吹打细胞悬浮在过滤器的顶部。</li>
	<li>将10毫升的 DMEM 再次添加到每个 mac 管中, 通过血清学吸管, 将剩余的细胞转移到50毫升离心管顶部的细胞过滤器上。</li>
	<li>使用血清学吸管, 用另外10毫升的 DMEM 低培养基清洗细胞过滤器。</li>
	<li>离心机细胞悬浮7分钟在 300 x g 在 RT 和放弃上清。</li>
	<li>并用重悬细胞在5毫升预热前内皮基介质 (循证医学)-2 微血管 (MV) 培养基 (龙沙) 补充与笔/链球菌/ampho 在同一50毫升离心管。</li>
	<li>平板细胞悬浮在 T-25 细胞培养瓶预先涂覆1.5% 明胶磷酸盐缓冲盐水 (PBS) 在37°c 与 5% CO2。</li>
	<li>孵育细胞为24小时在37°c 和 5% CO2, 仔细洗涤细胞两次与大约5毫升 PBS, 并且增加5毫升新鲜的媒介。</li>
	<li>培养细胞在37°c 和 5% CO2直到80-90% 融合 (通常 5-7 天) 以 48 h 间隔中等更新。</li>
</ol>3. 血管内皮细胞的分类
注意: 尽量避免在任何时候丢失细胞,例如通过限制染色程序在单个试剂管中孵化细胞。
<ol><li>分离细胞与1毫升的 accutase (大约10分钟), 并添加4毫升预热 (37 °c) EBM-2-MV 培养基。</li>
	<li>使用自动单元计数设备 (范围 10-25 µm) 确定单元格计数。</li>
	<li>离心细胞悬浮4分钟, 250 x g 在 RT 和放弃上清。</li>
	<li>根据细胞计数 (5 x 106/毫升), 并用重悬细胞 (1x PBS, 2.5% 牛血清白蛋白 [BSA], 5 毫米 EDTA pH 值 8.0, 10 µM Y-27632)。</li>
	<li>根据细胞计数/流计-缓冲容积添加可操作的染色抗体: CD31-FITC (100 µL/毫升 = 1/10), CD144/vascular 内皮 (VE)-钙粘蛋白-PE (100 µL/毫升 = 1/10), CD105-APC (25 µL/毫升 = 1/40), 混合和孵化为7分钟在 RT 保护免受光;轻轻地拍打和孵化8分钟 (总孵化时间为15分钟)。</li>
	<li>使用血清学吸管在标记的细胞悬浮液中加入2毫升的流化液缓冲器。</li>
	<li>离心机为4分钟, 250 x g 在 RT 和放弃上清。</li>
	<li>通过添加2毫升的内流器缓冲液洗涤两次, 然后在 RT 上进行4分钟的离心处理, 并将其丢弃上清。</li>
	<li>并用重悬细胞在500µL 预预热的 EBM-2-MV-pen/strep/ampho 和转移细胞到转位仪分拣仪器。<ol>
<li>如果该仪器冷却, 内皮细胞在分拣过程中很快就会死亡。因此, 在 RT 中进行血管内皮细胞的分类, 并立即将采集到的细胞转移到37摄氏度孵化器。 注: 该仪器通常 unsterile!</li>
	</ol>
</li>
	<li>将三阳性细胞直接放入细胞培养皿中 (例如, 24-在一夜之间用1.5% 明胶-PBS) 填充0.5 毫升预热后的新鲜培养基 (EBM-2-MV-pen/strep/ampho)。 注: 使用显微镜进行分拣后检查细胞悬浮。如果细胞聚集在一起, 尝试通过慢慢混合或添加培养基来获得细胞培养皿中的均匀细胞分布。</li>
	<li>培养细胞直到 90-100% 融合以 48 h 间隔的培养基更新和扩大细胞到所需的培养皿尺寸 (24 井板→12井板→ 3.5 cm 盘→ T-25 → T-75)。</li>
</ol>4. 纯内皮细胞的收获和鉴定
<ol><li>收获细胞在 90-100% 融合和特征他们由适当的方法 (例如,化学, 定量聚合酶链反应 (qPCR), 或内皮细胞功能化验7,8)。</li>
	<li>选择用任何一种表征方法分析孤立细胞。</li>
</ol>

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作者声明他们没有竞争的兴趣。

主要有三种不同的方法被使用到目前为止, 以分离纯净和可行的 NECs 和 TECs 从人的固体组织, 即 (1) 磁浓缩, (2) 激光显微切割, (3) 对 EC 分数的分类。在大多数出版物中, 磁通过机械解体产生单个细胞悬浮后细胞的富集被使用11,12,13。例如, 磁纯化的人皮肤微血管 EC (HDMEC) 的电子选择素抗体后, 肿瘤坏死因子 (TNF)-α治疗从新生儿包皮13已报告, 从神经胶质瘤分离的组织文摘, percoll 梯度和选择使用 anti-CD31/CD105 和 VE 钙粘蛋白抗体12, 或 anti-CD105 抗体从人乳腺癌11。使用激光显微切割或外地资产管制的协议-排序已不太频繁报告。以激光显微切割为例, 通过解剖9后的细胞分析, 确定人结直肠癌产生的可能的泛肿瘤内皮细胞标记物 (tem)。成功地证明了利用 anti-CD31-antibodies 对未分化人类胚胎干细胞的 EC 分数进行分类的方法14。
这些方法有不同的优缺点, 必须加以考虑。以磁为基础的方法的优点是, 不需要精心制作的设备, 任何时候都可以进行选择, 而细胞富集速度快 (约15分钟), 相对于系统分类 (大约1小时)。缺乏基质的时间较长可能诱发 EC 细胞死亡的脱落凋亡。因此, 将 Y-27632 激酶抑制剂添加到流化酶缓冲器中, 以防止细胞脱落凋亡15。
磁富集的缺点是, 为了达到99% 以上的纯度, 需要多轮选择。此外, 细胞的培养需要充实之间的细胞恢复, 这增加了文化的年龄支持文化诱导的文物。激光显微切割的优点是, 细胞可以立即使用, 这减少了文化造成的文物, 但包括由细胞污染的相邻组织区域的风险10。此外, 每个实验室都没有建立显微解剖。基于分类的方法的缺点包括与激光显微切割相似, 这些设备是精心设计和成本密集型的。因此, 这种设备可能在任何时候都无法访问。在临床环境中, 如果有兴趣组织的可用性不能确切地计划, 这可能会增加一个进一步的劣势。
然而, 这种分类的主要优点是, EC 分数可以用多个抗体并行标记。因此, 可以采用严格的门控策略, 确保高纯度。根据经验, 不需要对 EC 分数进行重新排序, 与以前的基于 mac 的协议相比, 必须使用多轮选择。这导致了从六周 (mac 方法) 到纯度的培养时间明显减少到三周, 在该方法中, 从而使细胞的生存能力得到改善, 从而可以长时间的分析窗口。最后, 采用基于流式分析的分类策略, 可以提高整体隔离成功率 (增加 12.5%)。总之, 在组织消化联合机械和酶组织消化后, 采用3种抗体平行的基于 TECs 的分类策略, 有许多优点, 建立了该协议作为分离的选择方法从人大肠癌和结肠 NECs。

肿瘤微环境被定义为肿瘤细胞与肿瘤基质的密切相互作用, 它由内皮细胞 (EC)、毛细血管、成纤维细胞、平滑肌细胞或免疫细胞等组成。这些细胞间的通讯可以由分泌因子 (例如,血管生成因子, 细胞因子), 由细胞外基质或直接细胞细胞接触驱动。基质隔间可以促进或抵消肿瘤的启动或进展, 具体取决于所建立的特定的病程。
肿瘤与血管系统连接的能力是疾病进展和转移的关键。该血管系统允许肿瘤主要获得氧气和养分的传递, 以及清除废物产品1,2,3。EC 构成了血管的内表面, 因此是积极参与这一过程的重要的细胞成分。众所周知, 肿瘤内皮细胞 (泄漏) 与相应的正常内皮细胞 (NEC) 有不同的特点, 如血管树紊乱的层次结构, 血管的, 或减少的成熟, 以减少数量的仅与 EC4松散连接的毛细血管/壁画单元格。
因此, TECs 是研究癌变的宝贵的细胞工具。TECs 主要被考虑促进肿瘤的生长和进展3。因此, TECs 可作为生物传感器, 用于监测和鉴定引发、促进或抵消肿瘤发生的致病过程。此外, 它们是临床治疗的目标5。因此, 孤立的 TECs 和相应的 NECs 也可以作为工具, 以了解反应机制或抗血管生成治疗。
在过去, 我们开发了一个协议来隔离这些单元格6,7 , 并确定 TECs 不仅与 NECs 不同, 而且根据它们从8中获得的期间而有所不同。通过这种方法, TECs 在某些巴掌就可以通过分泌抗血管生成肿瘤抑制蛋白 (如 SPARCL1) 积极对抗肿瘤的生长和进展。这表明, TECs 正积极促进建立 prognostically 有利的人大肠癌 (CRC)8。
以前的研究试图将人类 TECs 与实体肿瘤隔离开来。这些研究的一个重要目标是,例如,鉴定新的肿瘤内皮细胞标记 (方法)9。采用激光显微切割后立即使用 TECs 的策略, 以避免培养中的科技型的改变或丧失。然而, 后续研究发现, 被污染的壁画细胞的人口是这种方法的严重缺点10。我们的实验室是第一个开发一个协议, 允许隔离的纯, 可行的 TECs 从人类 CRC 患者6,7。选择了 TECs 多磁选 (mac) 轮的方法, 保证了分离的 EC 培养物的高纯度。然而, 这种方法需要一个相对较长的耕种期 (平均6周), 这增加了文化诱导的文物的风险。因此, 在下一步, 目的是减少手术之间的种植时间和收获的第一纯文化。为了实现这一目标, 开发了一种改进的协议, 采用了基于机械和酶结合的初始肿瘤组织离解, 其次是荧光活化细胞分选-三标记 EC 的分类。这减少了平均三周的隔离时间, 导致了纯科技和 NEC 文化, 并提高了功能研究的可行性。从高成功率的人体组织中分离出纯可行的科技和 NEC 文化, 可为个体化治疗方案的发展开辟新的病人特定药物检测途径。以下各段详述了隔离方法。

<table><tbody><tr><td>HBSS 1x</td><td>Gibco by Life Technologies</td><td>14175-053</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco by Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>amphotericin B</td><td>Gibco by Life Technologies</td><td>15290-026</td><td></td></tr><tr><td>Scalpels, disposable</td><td>Feather</td><td>No. 23</td><td></td></tr><tr><td>gentleMACS octo dissociator</td><td>Miltenyi Biotec</td><td>130-095-937</td><td></td></tr><tr><td>gentleMACS C-tubes</td><td>Miltenyi Biotec</td><td>130-093-237</td><td></td></tr><tr><td>human tumor dissociation kit</td><td>Miltenyi Biotec</td><td>130-095-929</td><td></td></tr><tr><td>DMEM</td><td>Gibco by Life Technologies</td><td>21969-035</td><td></td></tr><tr><td>EGM-2-MV BulletKit</td><td>Lonza</td><td>CC-3202</td><td></td></tr><tr><td>Cell strainer, 100 &amp;micro;m</td><td>Falcon</td><td>352360</td><td></td></tr><tr><td>StemPro Accutase&amp;nbsp;</td><td>Gibco by Life Technologies</td><td>A11105-01</td><td></td></tr><tr><td>Cell counter, automated</td><td>Coulter Counter</td><td>Z1</td><td></td></tr><tr><td>Y-27632</td><td>Sigma-Aldrich</td><td>Y0503</td><td></td></tr><tr><td>CD31-FITC</td><td>BD Pharmingen</td><td>555445</td><td></td></tr><tr><td>CD144/VE-cadherin-PE</td><td>BD Pharmingen</td><td>560410</td><td></td></tr><tr><td>CD105-APC</td><td>BD Pharmingen</td><td>562408</td><td></td></tr><tr><td>gelatin from bovine skin</td><td>Sigma-Aldrich</td><td>G9391</td><td></td></tr><tr><td>FACS-Sorting device</td><td>Beckman Coulter</td><td>MoFlo XDP</td><td></td></tr></tbody></table>

isolation of human endothelial cells from normal colon and colorectal carcinoma - an improved protocol

从人类肿瘤中分离出的原代细胞是鉴别致病机制的重要工具, 有助于疾病的发展和进步。特别是, 内皮细胞 (EC) 构成血管的内表面, 直接参与氧气的输送, 营养供应, 并清除废物的产品, 以和从肿瘤, 从而显著参与了肿瘤的构成微环境。肿瘤内皮细胞 (TECs) 可作为肿瘤与基质细胞间通讯建立的瘤内微环境的细胞生物传感器。TECs 也作为治疗的目标。因此, 在文化中, 这些细胞允许研究反应机制或抗血管生成疗法。最近发现, 从人类大肠癌 (CRC) TECs 分离出的记忆样效应的基础上, 他们的具体来源。此外, 这些 TECs 积极协助建立一个具体的, 由不同的因素分泌。例如, TECs 在 prognostically 有利的 Th1-TME 分泌的抗血管生成抑癌因子分泌蛋白, 酸性和丰富的半胱氨酸样 1 (SPARCL1)。SPARCL1 调节血管的稳态, 抑制肿瘤细胞的增殖和迁移。因此, 从人类实体肿瘤中分离出来的纯净、可行的 TECs 的培养, 是研究血管系统在肿瘤发生中作用的重要工具。本文介绍了一种新的从正常结肠和 CRC 分离初级 EC 的最新协议。该技术是基于机械和酶组织消化, immunolabeling 和荧光活化细胞分类 (资产管制)-排序的三重阳性细胞 (CD31, VE 钙黏蛋白, CD105)。通过该协议, 可行的科技或正常内皮细胞 (NEC) 的文化可以从结肠组织分离的成功率为 62.12%, 当受到的分类 (41 纯欧共体文化从66组织样本)。因此, 本议定书提供了一个强有力的方法, 以隔离人类 EC 文化从正常结肠和 CRC。

本文介绍了从人类 CRC 中分离的 NEC 和 enzymatical 组织分离, 接着是随后的 CD31/CD105/VE-cadherin-driven--排序在这里描述 (图 1a)。此协议表示改进后的协议, 缩短了时间窗口, 直到第一次收获纯、可行的内皮细胞, 而不是以前基于 mac 的协议7。
NEC 和科技从人类 CRC 中分离出来, 使用以下步骤: (1) 通过机械和酶组织的分离和生长, 将这些细胞分解为 80-90% 融合 (图 1), (2) 欧共体的三重标记, 产生单个细胞悬浮。通过 CD31/CD105/VE-cadherin fluorochrome 耦合抗体, (3) 对各自三重阳性细胞 (图 2A) 进行分类。值得注意的是, 成功的隔离程序和细胞存活的关键步骤是迅速将手术标本与隔离程序 (单细胞悬浮 < 切除后1小时) 分开。使用 "资产管制" 分类, 可以隔离12500个 (n = 12) 和 17800 NEC (n = 12,图 2b, 左) 的平均值 (图 3.6B, 右)。随后, 细胞扩大到 T-75 (称为通道 1), 或者收获或进一步处理以进行分析。注意, 使用以前的基于 mac 的协议, 实现了平均隔离成功 49.6% (n = 58 纯 TECs 从 n = 117 患者8) 与纯净的欧共体文化可利用在平均6星期在手术以后。使用此处描述的新的基于流式程序的协议, 在手术后平均3周获得了第一个纯 ec 文化, 隔离成功率为 62.1% (n = 41 纯 ec 文化, 从 n = 66 单细胞悬浮液中获得)。因此, 隔离率增加了 12.5%, 同时实现了从6周到3星期的种植时间的减少。这种减少的耕种时间导致改善细胞的生存能力和延长的寿命伴随着较少暴露于诱导潜在的文化依赖的文物的既定文化。
EC 纯度的特性首先由 CD31-cytochemistry (图 3A) 进行。如果这些区域性没有 CD31-negative 单元格 (图 3A、TEC 和 NEC), 则它们通过反向转录定量聚合酶链反应 (RT-qPCR) (图 3B), 受到更详细的细胞类型化。细胞型特定底漆为 EC (CD31, CD105, VE 钙黏蛋白, 血管性血友病因子 [vWF]), 白细胞 (CD45), 上皮细胞 (CK)-20), 平滑肌细胞/成纤维细胞 (desmin) 被使用 (图 3B)。使用这种基于 qPCR 的单元格类型, 可能会检测到 0.1-2% 范围内其他单元格 EC 区域性的潜在污染, 这取决于单元格的种类, 这可以探测到8。
值得注意的是, 我们以前报告在科技文化中 vWF 蛋白表达的优先损失与相应的 NEC 文化7相比。这些发现可以通过新建立的隔离协议 (图 3C、平均 Ct 损失 NEC = 0.687、 p = 0.173、配对 t 测试) 来确认。成功通过这些质量控制的文化测试了支原体感染, 并随后用于进一步的实验。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57400/57400fig1.jpg"> 图 1: 从人正常结肠和 CRC 中分离出纯内皮细胞.(A) EC 隔离过程的基本步骤流程图。(B) 脂肪组织 (黄色)、其他非肿瘤部位或潜在坏死组织部分 (褐色) 从手术标本 (左、中板) 中取出, 肿瘤在组织离解前被分解成 2-3 毫米侧长的立方体 (右面板)。(C) 在细胞培养皿中, 在组织离解 (左面板) 上孵化出24小时的单细胞悬浮液。随后, 用 PBS (中间板) 进行温和洗涤去除非黏附细胞, 细胞在融合 (右面板) 前生长到 80-90%。下板 (C) 的图像是通过相对比显微镜获得的。<a href="https://cloudflare.jove.com/files/ftp_upload/57400/57400fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57400/57400fig2.jpg"> 图 2: 平均 3.6-5.6% 的单细胞悬浮分离的人 CRC 患者和排序的外地观察组是三重阳性 EC.(A) NEC 和 anti-CD31 使用三种标记的细胞进行了分类--CD105 和 VE 钙黏蛋白抗体。(B) 12500 TECs (n = 12) 和 17800 NECs (n = 12) 的平均值可以与人正常结肠或 CRC 分离, 方法是使用该分类器 (CD31, CD105 和 VE 钙黏素阳性细胞, 左面板), 代表平均3.6 和 5.6% (右面板) 的总细胞被雇用的人口。<a href="https://cloudflare.jove.com/files/ftp_upload/57400/57400fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57400/57400fig3.jpg"> 图 3: NEC 和科技从人类 CRC 表达典型的内皮细胞标记.正常结肠内皮细胞 (NEC, n = 10) 和肿瘤内皮细胞 (科技, n = 10) 从人类 CRC 是 CD31 (ec 标记)-, CD105 (ec 标记)-, VE 钙粘蛋白 (ec 标记)-, vWF (ec 标记)-阳性和 CK20 (CRC 细胞标记)-, desmin (成纤维细胞/SMC 标记)-, CD45 (白细胞标记)-阴性, 由 (A) CD31-immunocytochemistry (阳性细胞 = 红色) 和 (B) RT qPCR (虚线下面的数据点是检测到的样本为负值)。(C) vWF 表达式, 由来自同一患者的相应的科技/NEC 样本的 RT qPCR 确定。<a href="https://cloudflare.jove.com/files/ftp_upload/57400/57400fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol at JoVE.com

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

肿瘤内皮细胞是肿瘤微环境和病程的重要决定因素。本文介绍了一种从人大肠癌和正常结肠分离纯化和存活的内皮细胞的协议, 用于药物检测和病机研究。

正常结肠和结直肠癌患者内皮细胞的分离--一种改进的协议

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

isolation-human-endothelial-cells-from-normal-colon-colorectal

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

15.2K 次观看.  Friedrich-Alexander University of Erlangen-Nuremberg. 肿瘤内皮细胞是肿瘤微环境和病程的重要决定因素。本文介绍了一种从人大肠癌和正常结肠分离纯化和存活的内皮细胞的协议, 用于药物检测和病机研究。

视频: Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

科研

癌症研究

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

人脐血内皮祖细胞的分离

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

发育生物学

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

生物学

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

医学

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

从健康志愿者和它们的迁徙可能通过血清中的影响心脏手术后的内皮祖细胞的分离

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

免疫与感染

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

小鼠原代肺内皮细胞的分离

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

鼠标冠状动脉内皮细胞的分离

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

从不同的脂肪库分离和鉴定血管内皮细胞，用于下游应用

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples

The availability of cells isolated from healthy and diseased tissues and organs represents a key element for personalized medicine approaches. Although biobanks can provide a wide collection of primary and immortalized cells for biomedical research, these do not cover all experimental needs, particularly those related to specific diseases or genotypes. Vascular endothelial cells (ECs) are key components of the immune inflammatory reaction and, thus, play a central role in the pathogenesis of a variety of disorders. Notably, ECs from different sites display different biochemical and functional properties, making the availability of specific EC types (i.e., macrovascular, microvascular, arterial, and venous) essential for designing reliable experiments. Here, simple procedures to obtain high-yield, virtually pure human macrovascular and microvascular endothelial cells from the pulmonary artery and lung parenchyma are illustrated in detail. This methodology can be easily reproduced at a relatively low cost by any laboratory to achieve independence from commercial sources and obtain EC phenotypes/genotypes that are not yet available.

Although challenging, the isolation of pulmonary endothelial cells is essential for studies on lung inflammation. The present protocol describes a procedure for the high-yield, high-purity isolation of macrovascular and microvascular endothelial cells.

从肺源性样品中分离和纯化微血管和大血管内皮细胞

The availability of cells isolated from healthy and diseased tissues and organs represents a key element for personalized medicine approaches. Although ...